Mechanism of regulation of the Helicobacter pylori Cagβ ATPase by CagZ

The transport of the CagA effector into gastric epithelial cells by the Cag Type IV secretion system (Cag T4SS) of Helicobacter pylori (H. pylori) is critical for pathogenesis. CagA is recruited to Cag T4SS by the Cagβ ATPase. CagZ, a unique protein in H. pylori, regulates Cagβ-mediated CagA transport, but the underlying mechanisms remain unclear. Here we report the crystal structure of the cytosolic region of Cagβ, showing a typical ring-like hexameric assembly. The central channel of the ring is narrow, suggesting that CagA must unfold for transport through the channel. Our structure of CagZ in complex with the all-alpha domain (AAD) of Cagβ shows that CagZ adopts an overall U-shape and tightly embraces Cagβ. This binding mode of CagZ is incompatible with the formation of the Cagβ hexamer essential for the ATPase activity. CagZ therefore inhibits Cagβ by trapping it in the monomeric state. Based on these findings, we propose a refined model for the transport of CagA by Cagβ.


On line 159 the authors reference the ATP binding site at the inter-subunit interface and reference Figure 1h. It is not clear where the proposed ATP binding site is in this
17. In Figure 1g the authors intended to show the lack of interactions between adjacent AAD domains within the hexameric structure. This is not clearly shown. In fact, it looks like the authors even highlight a single interaction. It would help to demonstrate the distances observed between residues.
18. In Figure 1d the authors reference the positions of the AADs in the hexamer and note that protomers A, B, and C have different orientations than D, E, and F. This is not apparent in the figure.

On line 187 the authors reference a loop that constricts the central channel.
Although a loop is represented in Figure 1c (denoted as the α5-α6) it is not clear if this is the loop being referred to. It would be beneficial to the reader to clarify this. 20. Lines 192-197 are speculative and should be moved to the discussion.

TrwB. It would be helpful if the authors included an image showing the electrostatic character of TrwB for comparison.
22. On lines 225, the authors reference an experiment that they conducted using three different constructs of the AAD domain. It is not clear to the reader why these three constructs were selected.

On line 234 the authors describe the two molecules in the asymmetric unit as being 'essentially identical'. The authors should provide an RMSD for the two copies.
24. On line 248 the authors describe the total buried surface area in the Cagβ-ADD-CagZ complex but do not describe how it was determined. A computational program should be cited.

Line 255 describes contributions from two residues (L196 and L198) to the Cagβ-
CagZ interface. The authors should specify which proteins these residues are from to avoid confusion.
27. Reference 53, as cited on line 360, does not refer to the H. pylori type four secretion system. Figure 6, the authors present ATP hydrolysis data to enumerate the hydrolytic activity of Cagβ. While error bars are represented on the graphs, it is not described in the methods or figure legend how the errors were calculated.

In
We thank the reviewers for their constructive critics and suggestions. We have carried out additional experiments to address some of the major concerns, including mass photometry experiments to confirm the conversion of Cagβ between the monomeric and hexameric states, and mutational analyses to support our assignment of the ATPase active site residues. In response to the comments of Reviewer 3 regarding the poor quality of some of the ITC data, we have replaced these data with new ones. We have also reprocessed all the ITC data and changed the presentation of the errors in Table 1, which led to some small changes to the numbers but the conclusions remain the same. The following are point-by-point response to the reviewers' comments.

Reviewer #1 (Remarks to the Author):
In this manuscript, Wu et al. describe crystal structures of the Helicobacter pylori ATPase Cagbeta and a complex formed between a fragment of Cagbeta and CagZ, a protein that interacts with Cagbeta and regulates the translocation of the oncoprotein CagA through the type 4 secretion system into gastric epithelial cells where it dysregulates cell signaling in numerous ways that can lead to peptic ulcer diseases and gastric cancer. The authors show that full-length Cagbeta forms a hexameric ring structure and that the Cagbeta fragment bound to CagZ forms a 1:1 complex that is incompatible with hexameric formation. Furthermore, they performed activity assays to show that the ATPase activity of Cagbeta is inhibited in a dose-dependent manner by CagZ, but not a mutant of CagZ that does not bind to Cagbeta. The data is quite compelling and its presentation is generally fine. A few major points of concern follow: Response: We thank the reviewer for these positive comments on the importance of this work and the quality of the manuscript.
1. Much of the authors' conclusions rest on the structure of the Cagbeta fragment-CagZ complex and the size exclusion chromatography analysis of full-length Cagbeta in the presence and absence of CagZ. In this crystal structure, there are two notable features: first, the Cagbeta is a fragment and, thus, not the natural full-length Cagbeta protein but an arbitrarily truncated portion; second, the C-terminal tail of CagZ in this structure adopts a conformation that is different from that of the apo CagZ structure and, moreover, makes numerous interactions with the Cagbeta fragment, which also undergoes conformational changes relative to its apo structure. Whether these conformational changes in both proteins and the resulting intermolecular interactions are caused by crystallization or not is unclear. Some solution biophysics could help here. There are numerous options, but performing a hydrogen-deuterium exchange-mass spectrometry experiment with the proteins in the crystal structure, as well as with full-length Cagbeta, would show both the interfaces gained by the interactions of these proteins and the interfaces lost in the proposed CagZ-mediated transition of full-length Cagbeta from hexameric to monomeric forms. These HDX-MS experiments, as well as additional analytical ultracentrifuge experiments, would also greatly corroborate the SEC data suggesting that CagZ transitions Cagbeta from hexamer to monomer (SEC is not the highest fidelity readout of oligomeric state).
Response: We agree with the above comments and thank the reviewer for the insightful suggestions. HDX-MS is a highly specialized technique, which we cannot do on our own. It would cause much delay even if we could manage to find a collaborator to do these experiments. A crystal structure of full length cagβ in complex with CagZ would be great to have. In fact, we have tried co-expression and co-crystallization of Cagβ1 (full-length of the soluble region) with CagZ. Unfortunately, all attempts of crystallization of this complex failed. It is likely that, while the AAD is stabilized in the Cagβ1/CagZ complex, the NBD and the NBD-AAD interdomain hinge become flexible due to lack of the stabilization effect of the hexameric assembly, rendering the complex refractory to crystallization.
Regarding the conformational changes of CagZ upon binding to Cagβ, in the apo-CagZ structure published 2004 1 , the authors predicted that the disordered C-terminal tail and the charged patch of the CagZ surface participate in protein/protein interactions 1 . Our structure of the complex is consistent with these previous analyses. In addition, our extensive mutational data support the critical role of the CagZ tail in binding to Cagβ. As discussed in the manuscript, there is no evidence suggesting that the conformational changes to the AAD of Cagβ in the structure make major contributions to the binding to CagZ or regulation of Cagβ in general. We therefore don't assign significance to these changes.
We agree with this reviewer that another method to confirm that the transition of Cagβ from the hexamer to monomer upon binding CagZ is useful. In the revised manuscript, we address this point by using mass photometry, a new technique that can accurately measure the molecular weight of proteins 2,3 . The results show that (revised Fig. 5d) Cagβ1 at 1.80 µM is mostly hexameric, with ~74% of protein showing a measured molecular weight of ~408 kDa, very close to the theoretical molecular weight of the hexamer of ~402 kDa. The hexamer population decreased to 22% when the protein concentration was lowered to 0.06 µM, meanwhile the monomeric species (~70 kDa, close to the theoretical molecular weight of the monomer of ~67 kDa) became more prominent. The sample containing both CagZ and Cagβ1 showed molecular weight of ~88 kDa, consistent with the theoretical molecular weight of the 1:1 complex of ~90 kDa. These new data provide strong support for our monomer to hexamer transition model.

The authors' structure of full-length Cagbeta shows that its Walker A motifs responsible
for ATP binding and hydrolysis are formed within a single Cagbeta protomer (in "cis"), rather than through the participation of residues from neighboring protomers (in "trans") that is common to many Cagbeta homologs. This begs the question: why would CagZmediated trapping of a monomeric Cagbeta state inhibit ATP hydrolysis if a monomer still contains a whole Walker A motif? That is, what are the CagZ-driven changes, conformational or otherwise, in the Cagbeta Walker A motif that explain the related dysfunction of the ATPase I? Again, HDX-MS could indicate conformational and/dynamic changes in the Cagbeta Walker A motifs induced by CagZ.
Response: This is an important point, which we admit that we do not fully understand at present. As mentioned in the paper, most if not all the ATPases in this family function as hexamers, with the catalytic site residing at the inter-subunit interface. We reasoned that one important role of the hexameric interface is to help the Walker A and Walker B motifs adopting the catalysis-competent conformation. We agree that the arginine finger in cis in Cagβ is unusual and warrants further investigation. We therefore mutated R241 (R241A and R241K), the putative arginine finger residue and tested the effects of the mutations on the ATPase activity. The results show that these mutations completely abolish the ATPase activity, confirming that R241 is critical for catalysis. Furthermore, we mutated key residues in Walker A (K244A) and Walker B (E551A and E551Q). These mutations also abolish the ATPase activity. These new data, included in revised Figure 6a, support our model on the catalytic mechanism. Some minor comments follow: 1. There are a number of instances in which the order of figures and/or figure panels appear out of order with the text (e.g., figure 2 is described in text after the early panels of figure 1 but before the latter panels of figure 1; figure 7b is described before 7a).
Response: We thank the reviewer for pointing out these errors. We have made corrections by changing the order of the figures and the text.

Line 204: "biochemical assays" is vague.
Response: We have replaced this term with the specific techniques used (Size-exclusion chromatography and cross-linking assays).

Line 208: "superstable" -is this a technical term?
Response: We agree that this is not an accurate term. We now simply describe the hexamer as not very stable.

Reviewer #2 (Remarks to the Author):
This manuscript reports an exciting advance in the T4SS field concerning the structural definition of the Cagb coupling protein and effects of the CagZ adaptor Istructure and catalytic activity. The Cagb structure is only the second X-ray structure of the T4CP components of T4SSs, which allowed for detailed comparisons between Cagb and the current structural prototype for T4CPs, TrwB. The T4CPs are essential for translocation of DNA and protein substrates through most T4SSs, therefore, it is critical to know their architectures and effects of modulatory proteins such as CagZ. The apostructure of CagZ was previously reported, and here the authors show that CagZ undergoes a profound conformational change when bound to the all-alpha-domain (AAD) of Cagb. Previous studies have shown that T4CP AADs are important receptor domains for secretion substrates, and here the authors propose that CagZ coordinates binding of the secretion substrate CagA with the Cagb T4CP for translocation through the Cag T4SS.

CagZ locks Cagb into the monItate and also blocks its ATPase activity, suggesting that CagZ binding renders Cagb inactive. Their findings prompt a model in which the binding of CagA to the CagZ-Cagb complex results in dissociation of CagZ, which in tuIes Cagb hexamer formation and catalytic activity as a prerequisite for Cagb-mediated unfolding and translocation of CagA. Overall, the work is well-described and appears technically sound although this reviewer is not an X-ray crystallographer. It represents a major advance in the field insofar as there is no prior information describing in atomic detail the structural effects of an adaptor on the nucleotide binding domain of a T4CP. There are a number of grammatical flaws that can be easily rectified. As mentioned below, the authors should consider their model in the context of the recent CryoET structure of the Cag machine in the H. pylori envelope, which shows densities potentially corresponding to the Cagb hexamer at the base of the central channel. Other issues are minor.
Response: We thank this reviewer for these positive comments.

Pg. 5, L. 139 Walker. Fix other grammatical errors throughout.
Response: Thanks for spotting this error. We have thoroughly checked the manuscript to correct this and other errors.

Pg. 6. L. 150. This isn't evident from the presented structures, which isn't that important, but if the authors want to highlight this point, a different color scheme should be used for the C-terminal domain.
Response: We agree and therefore have deleted this sentence.

Pg. 6. L. 155. Not evident since there's no side-by-side comparison in this figure.
There is a comparison in the suppl figure 1a,b, but this also does not reveal the size difference. Figure 1). 4. Pg. 7. L. 226. Since no information can be gained for residues 260-460, it is possible that these residues also bind CagZ even though the AAD also does.

Response: Sorry for this oversight. We have added a new panel showing the side-by-side comparison of the two hexamers to illustrate the size difference (Revised Extended Data
Response: Our structure of the AAD/CagZ complex shows that residues 297-476 constitute the AAD required for binding CagZ. Residues 260-460 are therefore indeed mostly the AAD. This question might be caused by our poor writing of this paragraph. We have made changes to it and hopefully the description has better clarity now. Fig. 4 requires a fuller description in the legend about the data shown.

5.
Response: Thanks for this suggestion. We have revised the figure legend accordingly. 6. Fig. 7. and Discussion. The model presented in Fig. 7 should be referred to early in the Discussion, even within the first paragraph. The model presents a flow of step-wise binding reactions that can be referred to throughout the Discussion.
Response: Thanks for these suggestions. We have added the reference to the model throughout the discussion.

The model is quite interesting, but needs to be reconciled with a couple of observations. First, a deltacagZ mutant lacks detectable CagA and, second, the CryoET structure shows densities consistent with a Cagb hexamer at the base of the inner ring. Relating to the deltacagZ phenotype, it isn't clear why cagZ binding to the Cagb monomer destabilizes CagA. Relating to the Cagb hexamer-IMC architecture detected by CryoET, this is assumed to represent the Cag machine in its quiescent state. If this is the case, it isn't obvious why Cagb assembles as the catalytically hexamer in the quiescent machine. Couldn't another model simply be that Cagb assembles as a hexamer but when docked onto the Cag machine it is catalytically inactive? CagZ could function as an adaptor by binding CagA and recruiting it to the AAD. Upon CagA-CagZ-Cagb binding, the CagZ -Cagb interaction
induces conformational changes in the Cagb hexamer that do not necessarily induce monomerization but instead induces structural changes necessary for dIf CagA, an event that stimulates Cagb catalytic activity and, accordingly, CagA unfolding and translocation. This model is simpler and more consistent with results of the CryoET studies and CagZ-CagA biochemical studies. Regardless of whether the authors like this latter model, they need to do a better job reconciling their findings with the observations mentioned above.
Response: We appreciate this reviewer for these insightful comments. The actual processes of T4SS assembly and CagA transportation are likely much more complicated than reflected by the few structures presented by us and others. As pointed out by this reviewer, it is indeed intriguing that lack of CagZ causes loss of CagA. We speculate that CagZ is required for keeping the inactive pool of Cagβ, which in turn is required for timely assembly of T4SS when CagA needs to be translocated.
The presence of the Cagβ hexamer in the presumably quiescent state in the CryoET structure is another interesting but not fully understood question. It is worthy pointing out that in the more recent high-resolution cryo-EM structure of T4SS encoded by the R388 plasmid, the TrwB/VirD4 ATPase is missing, indicating that it is not stably associated (Reference 53 in the manuscript). It is possible that the assembly, docking and activation of the ATPase hexamer is a multi-step process. The cryo-ET and cryo-EM structures may represent different intermediate structures. The ATPase may remain inactive even when the hexamer is docked to the base of the inner ring. The formation of the hexamer is necessary for the activation of the ATPase, but the activity is regulated by additional mechanisms that ensure it is only fully active when CagA engages Cagβ. This idea is consistent with the model suggested by this reviewer, where CagA may promote the ATPase activity by inducing a conformational change to Cagβ. In this case, it is however unlikely CagZ remains bound to the AAD of Cagβ, because its binding is not compatible with the formation of the hexamer based on our structures. That said, we cannot formally rule out the possibility that CagZ may bind the hexamer Cagβ in a different mode at present.
We have revised the discussion section to incorporate these points inspired by this reviewer.

Reviewer #3 (Remarks to the Author):
In the manuscript entitled "Mechanism of regulation of the Helicobacter pylori Cagβ ATPase by CagZ" Wu et al. detail the structural analysis of the H. pylori protein known as Cagβ, an ATPase associated with the H. pylori Cag type four secretion system (T4SS). The function of Cagβ is to aid in the transference of the oncogenic protein CagA, into host cells. In this manuscript, the authors detail two structures of Cagβ, one in a hexameric assembly and one in the monomeric form bound to the accessory protein CagZ. The analysis described here highlights a regulatory mechanism for CagZ that suggests it prevents premature oligomerization of Cagβ. The authors provide evidenceIs mechanism using isothermal titration calorimetry, ATPase activity assays, and size exclusion chromatography. Although the observation is noteworthy and the presented mechanism interesting, it should be noted that several critical pieces of data are missing from the manuscript. Coupled with a severe lack of clarity, it is suggested thaI this manuscript is not suitable for publication. Noted below are several issues that have been raised for this manuscript.
Response: We thank this reviewer for the constructive critics. We have extensively revised the manuscript and included new data, which we hope can address these concerns.

Major Issues
1. The authors make several claims about the binding site of ATP in Cagβ but do not present a crystal structure with ATP bound. In fact, most of the analysis concerning the ATP binding site is dependent on the placement of a SO4-2 ion in the active site. Without a structure of Cagβ bound to ATP, these comments are speculative. It is suggested that the authors work towards a co-crystal structure of Cagβ in complex with ATP, or a nonhydrolysable analog.
Response: We agree that a crystal structure with ATP or a non-hydrolysable analog would be a valuable addition. We indeed tried repeatedly but failed to obtain high quality crystals for solving such a structure. As an alternative, we carried out mutational studies of key residues in the catalytic site according to our structural analyses. The results show that mutations of R241 (catalytic base), K244 (Walker A) and E551 (Walker B) abolish the ATPase activity of Cagβ (revised Figure 6a). These new data lend strong support to our assignment of the catalytic site residues.  Response: Sorry for the vague description. We describe the hexamer of Cagβ as having a three-layered structure: the top layer is composed of AAD, the middle layer is NBD, and bottom layer is formed by the β-barrel. We agree the term "base layer" might be misleading. We have changed it to "bottom layer". We have labeled the three layers in revised Figure  1b Figure 1d to address this point. We chose residue 374 in each protomer of the AAD and residue 704 in each protomer of the NBD as the reference points to measure the distance between neighboring subunits. It is clear from the figure that the distances between neighboring AAD are quite different from one another. For example, the distance between AADs of subunits E and F (45.7Å) is much larger than that between subunits A and B (36.9Å). In contrast, the same analysis of the NBD shows that they are related to an approximate 6-fold symmetry. Therefore, the deviation from the 6-fold symmetry is largely due to shifts of AAD relative to NBD. The actual shifts of each AAD appear somewhat random and may be influenced by crystal contacts, and therefore we do not to describe them in detail.